Single-molecule, high-resolution imaging of protein-nucleic acid complexes using techniques such as electron microscopy (EM) and atomic force microscopy (AFM) provides invaluable information about the structure-function relationships of biological processes. A significant limitation to these techniques, however, is the inability to resolve the location of the nucleic acid within protein complexes. Electron spectroscopic imaging selecting for phosphorous coupled with image averaging has been used to characterize the DNA content of nucleosomes1; however, there are no methods that allow visualization of DNA within protein-DNA complexes at a single molecule level. Because both proteins and DNA are significantly charged and interactions between proteins and DNA result in charge neutralization, we reasoned that it may be possible to visualize the path of DNA inside protein-DNA complexes by high-resolution imaging of their electrostatic potential.
Electrostatic force microscopy (EFM) and Kelvin probe force microscopy (KPFM) have been used to image the electrostatic surface potential of a large variety of materials with high spatial resolution and sensitivity2. There are several different modes of EFM and KPFM. Generally, a modulated bias voltage (VDC+VAC sin(ωt)) is applied between the tip and sample. This bias generates an electrostatic force between the tip and the sample, which is the sum of three components3,4:
                              F          DC                =                              1            2                    ⁢                                                    ∂                C                                            ∂                z                                      ⁡                          [                                                                    (                                                                  Δ                        ⁢                                                                                                  ⁢                                                  ϕ                          TS                                                                    -                                              V                        DC                                                              )                                    2                                +                                                      V                    AC                    2                                    2                                            ]                                                          (        1        )                                          F          ω                =                  -                                                    ∂                C                                            ∂                z                                      ⁡                          [                                                (                                                            Δ                      ⁢                                                                                          ⁢                                              ϕ                        TS                                                              -                                          V                      DC                                                        )                                ⁢                                  V                  AC                                ⁢                                  sin                  ⁡                                      (                                          ω                      ⁢                                                                                          ⁢                      t                                        )                                                              ]                                                          (        2        )                                          F                      2            ⁢            ω                          =                              1            4                    ⁢                                                    ∂                C                                            ∂                z                                      ⁡                          [                                                V                  AC                  2                                ⁢                                  cos                  ⁡                                      (                                          2                      ⁢                      ω                      ⁢                                                                                          ⁢                      t                                        )                                                              ]                                                          (        3        )            where ΔφTS and ∂C/∂Z are the contact potential difference and capacitance gradient, respectively, between the tip and the sample. This force induces a vibration in the cantilever at the frequency of the AC bias (ω). In KPFM, a feedback loop is used to adjust VDC such that it compensates for ΔφTS, thereby nullifying Fω and generating a potential map of the surface; whereas, in EFM, there is no feedback voltage, and images are produced by monitoring the amplitude (and phase) of the vibration. Dual-frequency single-pass techniques, where the topography and the surface electrical potential are captured simultaneously have the highest sensitivity2,4,5. In fact, dual-frequency KPFM has been used to obtain images of DNA5 and transcription complexes6; however, no details about the protein-DNA complex were revealed.
Accordingly, there exists a need for improved methods, systems, and computer readable media for imaging a biological sample, including charged structures residing beneath the surface of the sample.